Identification and expression of a regeneration-specific homeobox gene in the newt limb blastema

Adult urodele amphibians are able to regenerate their limbs through the formation of a blastema, a growth zone of undifferentiated mesenchymal cells that arises locally at the plane of amputation. In an effort to define genes involved in pattern formation by the blastema, we isolated from a newt forelimb blastema library cDNA clones that identify a homeobox gene termed NvHbox 2. The amino acid sequence of the homeodomain is identical to that of the recently identified human HOX-4f gene (Acampora et al. 1989) and of the mouse Hox-5.5 (Dolle et al. 1989). NvHbox 2 is expressed in the limb blastema as a transcript of 3.4 kb that is not detectable in the normal limb. Analysis by RNAase protection demonstrates expression in limb and tail blastemas, but not in any of the adult tissues or organs tested. In the limb blastema NvHbox 2 was expressed in mesenchymal but not epithelial tissue. When matched and normalised samples of RNA from proximal (mid-humerus) and distal forelimb (mid-radius) blastemas were compared, the level of expression of NvHbox 2 was found to be 3- to 5-fold higher proximally. At two time points after injection of a proximalising dose of retinoic acid, the level of expression in a distal blastema was not increased in comparison to controls.